Studies toward the biological and molecular understanding of programmed cell death have recently been stimulated by the identification of genes and their products which regulate apoptosis. Apoptosis represents an active process of autonomous cell death that occurs in physiological or pathological conditions (Clarke et al., 1990, Anal. Embryol. 181:195-213; Boehmer, 1992, Immunology Today 13:454-458; Gougeon and Matagnier et al., 1993, Science 260:1269-1270; Thompson, 1995, Science 267:1456-1462). Pathways to trigger programmed cell death may vary in different cells, but the regulation of apoptosis is generally mediated by the inducer and suppressor signals initiated from the molecular cascade during apoptosis. For instance, some members of the tumor necrosis factor/nerve growth factor receptor gene family are capable of inducing apoptotic cell death by perturbation of these molecules (Trauth et al., 1989, Science 245:301-305; Itoh et al., 1991, Cell 66:233-243; Oehm et al., 1992, J. Biol. Chem. 267:10709-10715), suggesting that they could act either by initiating a death-inducing signal or by blocking the signals required for cell survival. In contrast, the protein encoded by the Bc1-2 gene can promote cell survival by interfering with pathways leading to apoptosis although Bcl-2 does not appear to influence cell cycle progression (Vaux et al., 1988, Nature 335:440-442; Hockenbery et al., 1990, Nature 248:334-346; Nunez et al., 1990, J. Immunol. 144:3602-3610). The recent identification of these genes and their products that regulate programmed cell death has contributed greatly to our understanding of the molecular mechanism of apoptosis, and also represents a new challenge in defining novel molecules involved in programmed cell death.
Apoptosis is accompanied by characteristic morphologic changes and the degradation of internucleosomal DNA (Kerr et al., 1972, Br. J. Cancer 26:239-257; Wyllie, 1980, Nature 284:555-556). Recent evidence indicates that, prior to the occurrence of morphological changes and death itself in the spontaneous or induced apoptosis, the cells undergo substantial alterations in both phenotypic and functional properties. These include activation of endonucleases (Duke et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:6361-6365; Barry and Eastman, 1993, Arch. Biochem. Biphys. 300:440-450; Peitsch et al., 1993, EMBO J. 12:371-377; Zhang et al., 1995, Cell Immunol. 165:161-167), the expression of molecular markers (Estus et al., 1994, J. Cell. Biol. 127:1717-1727; Fernandez et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:8641-8645), and a loss or increase in protein expression (Kishimoto et al., 1995, J. Exp. Med. 181:649-655; Casciola-Rosen et al., 1994, J. Exp. Med. 179:1317-1330; Ajmani et al., 1995, J. Exp. Med. 181:2049-2058). Although molecular alterations have been shown to be closely associated with apoptosis, little is known about their precise role in the process of apoptotic cell death.
The molecular alterations in apoptosis have not only been observed in cell membranes and the nucleus, but also in mitochondria. Mitochondrial DNA may not be fragmented in the apoptotic cell, while its nuclear DNA has been cleaved into fragments by endonucleases (Murgia et al., 1992, J. Biol. Chem. 267:10939-10941; Tepper and Studzinski, 1992, Cancer Res. 52:3384-3390). However, the abnormal ultrastructure of mitochondria and a reduction in mitochondrial membrane potential have been found in cells undergoing apoptosis (Vayssiere et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11752-11756; Zamzami et al., 1995, J. Exp. Med. 181:1661-11672; Steller, 1995, Science 267:1445-1449). The localization of Bcl-2 to mitochondrial membranes has implicated some functional influence of mitochondria on apoptosis although the Bcl-2 gene product is also found in the nuclear envelope and endoplasmic reticulum (Hockenbery et al., 1990, Nature 248:334-346; Krajewski et al., 1993, Cancer Res. 53:4701-4714). Using a human fibroblast cell line that lacks mitochondrial DNA, Jacobson et al., (1993, Nature 361:365-369) reported that neither the induction of apoptosis by growth factor withdrawal nor the antiapoptosis effect of Bcl-2 appears to depend on the activity of mitochondrial respiratory chain. Overexpression of Bcl-2, however, does enhance the mitochondrial membrane potential and rescues cells from apoptotic cell death (Hennet et al., 1993, Cancer Res. 53:1456-1460). Consistent with these findings, Zamzami et al. (1995, J. Exp. Med. 181:1661-1672) have recently shown that a reduction in mitochondrial membrane potential is an early irreversible event of lymphocyte apoptosis in vivo, and that some pharmaceutical agents capable of blocking early signaling pathways for apoptosis efficiently stabilize the values of mitochondrial membrane potential. These data have suggested the involvement of mitochondrial components in apoptotic cell death.
To identify the molecular markers for apoptotic cells, monoclonal antibodies were developed by immunizing mice with dying Jurkat cells. An antibody, designated anti-7A6, was found to react preferentially with cells undergoing apoptosis and not with normal cells. The antibody-defined molecule is a 38 kD protein localized to the membrane of mitochondria.
The monoclonal antibody can be used to distinguish apoptotic cells from normal cells, study the molecular mechanisms of apoptosis and diagnose samples from apoptosis-related diseases, to monitor the efficacy of therapeutic regimens, and to identify novel agents which induce or inhibit apoptosis. Reference is made to the article by Thompson, (1995, Science 267:1456-1462) which describes in detail the role of apoptosis in the pathogenesis and treatment of disease.